The present invention arose from the need to produce a so-called Boersch phase plate. However, the method developed by the inventors can also be used for the production of other multi-layer electrostatic lens arrangements.
One frequently employed method for the examination of the structure of for example biological-medical objects is transmission electron microscopy (TEM), with which a resolution in the range of less than 1 nm can be achieved. With this, structural details of an object in the range of nearly atomic dimensions can be depicted. Biological objects consist however predominantly of light elements like carbon, hydrogen and oxygen, so that the high-energy electrons, which are used for the formation of the image, are virtually not absorbed. For this reason no usable amplitude contrast comes into being so that the object in the image remains invisible in the case of an amplitude contrast measurement.
However, the phase of the electrons is slightly shifted. Therefore one refers to objects with this property as phase objects. They can therefore be made visible with a phase contrast electron microscope. For the generation of the highest possible phase contrast in the case of phase objects the phase of the non-scattered electrons (null beam electrons) is shifted by about 90°, in order to obtain a maximum phase contrast in the case of the subsequent superimposition of the phase-shifted non-scattered electrons with the scattered electrons in an interference pattern. The phase displacement of the null beam can in principle be realized by a phase plate.
In light microscopy the use of a phase plate for phase contrast microscopy is already realized in practice. For this purpose a so-called Zernicke phase plate in the form of a λ/4 platelet in the back focal plane of the objective lens is used.
A known method for a TEM works with the generation of phase contrast without phase plates. In this connection it is necessary to defocus the image in order to obtain structural information about the object. In order to cover the greatest possible range of space frequencies so-called defocus series (several images with variable defocusing) are recorded. This is very awkward and tedious. Further complicating matters in the recording of image series is the fact that the samples are frequently photosensitive and can only be loaded with a low electron dose without significant radiation damage. For this reason the signal-to-noise ratio in the images of a defocus series is low. Therefore the image quality is in great need of improvement. To the best of the inventors' knowledge this method constitutes the only actually practicable method for phase contrast electron microscopy.
Another solution for the generation of phase contrast according to the knowledge of the inventors which however up to now is still more or less theoretical is based on the usage of a phase plate for a TEM which produces the desired phase displacement of typically 90° between scattered and non-scattered electrons in the back focal plane of the objective lens of the transmission electron microscope.
However, the technical realization of a phase plate for a TEM is extraordinarily difficult, a fact which lies in the extremely small wavelength of the electrons in the magnitude of 10−12 m in comparison to the wavelength of visible light (4 . . . 7×10−7 m).
An embodiment which is in the research stage provides a thin carbon film which is positioned in the back focal plane of the microscope objective lens. A small hole with a diameter of about 1 μm is located in the middle of the carbon film, through which said hole the beam of the non-scattered electrons passes. The scattered electrons on the other hand pass through the carbon film and undergo an additional phase displacement in relation to the non-scattered electrons through the inner potential of the carbon film. Such a phase plate is also termed as a Zernicke phase plate. The usage of such a Zernicke phase plate has proved impracticable up to now due to the following difficulties.
Due to the inelastic scattering of the electrons in the carbon film the electron coherence is partially lost. Further the granularity of the carbon film leads to spatial fluctuations of the phase displacement. In addition the carbon film is damaged by the high-energy electrons. The spatial inhomogeneous contamination of the carbon film connected to the slight thickness of about 3×10−8 leads to electrical charges and uncontrollable phase displacements. Further the phase displacement can be controlled only by the thickness of the film. If said thickness changes, for example through contamination, the plate must be expensively removed and replaced by a new plate. In principle, setting of the phase displacement is not possible without replacement of the plate.
For these reasons the usage of a Zernicke phase plate for a TEM has proved to be impracticable.
Recently in Ultramicroscopy 99, 211 (2004) Lentzen proposed realizing a Zernicke phase plate with the help of a double hexapole aberration corrector in the imaging system. The proposed structure however is expensive and extremely cost-intensive.
A considerably better variant for a phase plate in the opinion of the inventors is the so-called Boersch phase plate. While it is true that it was already proposed in the year 1947 by Hans Boersch in “About the Contrasts of Atoms in the Electron Microscope”, Z. Naturforschung, 2a, 615-633, 1947; to the best of the inventors' knowledge the actual production of a Boersch phase plate however has not been managed to this very day. The extraordinary difficulty lies in the small dimension of the phase plate and its nevertheless complex structure. For this reason only more or less theoretical approaches are known from EP 0 782 170 A2 and WO 03/068399. For the sake of a better understanding the fundamentals of a phase contrast microscope will be described first.
The principle of phase contrast electron microscopy is shown in FIG. 1. In the case of a transmission electron microscope 1 an electron source 2 produces a high-energy electron beam 4 which is directed by a first and second condenser lens 5, 6 in order to screen the sample 8 for an upper objective lens. The scattered and non-scattered electrons are focused by an objective lens 9 and traverse the phase plate 20 in the back focal plane of the objective lens 9.
The null beam 10, that is, the non-scattered electrons are guided through an electric field 12 which is produced by a ring electrode 14. Through the electric field 12 the null beam electrons 10 undergo a phase displacement in comparison to the scattered electrons 16 which do not pass through the electric field.
After a first intermediate image 17 the electrons pass through an intermediate lens 18 and a projection lens system 19 in order to produce an image 22 of the sample for example on a film or a CCD chip 24.
In EP 0 782 170 A2 a proposal is now made for a phase plate for a phase contrast electron microscope. For the contemplated production a foundation made of a silicon substrate, covered on both sides with etch-resistant layers, is used. This layer arrangement is however only a subcarrier and not a component of the phase plate to be produced (compare FIGS. 6a through 6e in EP 0 782 170 A2). The structure of the phase plate takes place by means of the depositing of insulating and metallic layers as well as etching method. Finally however the silicon carrier with the etch-resistant layers must be completely removed in a two-stage process by means of etching of the highly sensitive phase plate. EP 0 782 170 A2 is silent about how this is to take place in detail. Also in other respects the production remains unclear in many points. However, the method is complicated by a multitude of depositing and etching steps as well as the removal of the auxiliary substrate. In addition a great probability exists that in the event of the removal of the auxiliary substrate the highly sensitive phase plate will be destroyed. For this reason the method appears to be at least little practicable.
However, an actual realization is not known.
In addition FIGS. 6a through 6e as well as the description on Page 7, lines 4 through 35 in EP 0 782 170 A2 only show the production of the central electrode of the phase plate. However, said electrode must be suspended in an aperture stop in the case of a Boersch phase plate. EP 0 782 170 A2 gives no information at all about how this should happen, so that no complete teaching on the production of a Boersch phase plate can be inferred from here.
Further WO 03/068399 describes a phase plate for electron microscopy. In this publication however, it is a matter of the non-mirror-symmetrical arrangement of the carriers of the electrode. This special geometry is described as advantageous for the reconstruction of image information using the Friedel symmetry. This phase plate consists of an annular electrode with an inside diameter of about 1 μm, through which the null beam is guided. The ring electrode is fastened for example via three webs to a mount. Apart from some preferred forms of the mounting device (compare FIGS. 1-3 of WO 03/068399) formed from the carriers the document does not specify its own production method. Reference is only made to EP 0 782 170 with regard to the details of the structure of the phase plate. For this reason the foregoing described disadvantages and problems are the same.
Although the Boersch phase plate was already proposed in 1947 by Hans Boersch, even including the teachings from EP 0 782 170 and WO 03/068399, to this day no Boersch phase plate that has been actually produced is known. To the best of our knowledge, the inventors have now succeeded in doing this for the first time with the production method proposed here.